The SIX2/PFN2 feedback loop promotes the stemness of gastric cancer cells.

BACKGROUND: The roles of the transcriptional factor SIX2 have been identified in several tumors. However, its roles in gastric cancer (GC) progression have not yet been revealed. Our objective is to explore the impact and underlying mechanisms of SIX2 on the stemness of GC cells. METHODS: Lentivirus infection was employed to establish stable expression SIX2 or PFN2 in GC cells. Gain- and loss-of-function experiments were conducted to detect changes of stemness markers, flow cytometry profiles, tumor spheroid formation, and tumor-initiating ability. ChIP, RNA-sequencing, tissue microarray, and bioinformatics analysis were performed to reveal the correlation between SIX2 and PFN2. The mechanisms underlying the SIX2/PFN2 loop-mediated effects were elucidated through tissue microarray analysis, RNA stability assay, IP-MS, Co-Immunoprecipitation, and inhibition of the JNK signaling pathway. RESULTS: The stemness of GC cells was enhanced by SIX2. Mechanistically, SIX2 directly bound to PFN2's promoter and promoted PFN2 activity. PFN2, in turn, promoted the mRNA stability of SIX2 by recruiting RNA binding protein YBX-1, subsequently activating the downstream MAPK/JNK pathway. CONCLUSION: This study unveils the roles of SIX2 in governing GC cell stemness, defining a novel SIX2/PFN2 regulatory loop responsible for this regulation. This suggests the potential of targeting the SIX2/PFN2 loop for GC treatment (Graphical Abstracts).

Identification of a novel lactylation-related gene signature predicts the prognosis of multiple myeloma and experiment verification.

Multiple myeloma (MM) is an incurable hematological malignancy with poor survival. Accumulating evidence reveals that lactylation modification plays a vital role in tumorigenesis. However, research on lactylation-related genes (LRGs) in predicting the prognosis of MM remains limited. Differentially expressed LRGs (DELRGs) between MM and normal samples were investigated from the Gene Expression Omnibus database. Univariate Cox regression and LASSO Cox regression analysis were applied to construct gene signature associated with overall survival. The signature was validated in two external datasets. A nomogram was further constructed and evaluated. Additionally, Enrichment analysis, immune analysis, and drug chemosensitivity analysis between the two groups were investigated. qPCR and immunofluorescence staining were performed to validate the expression and localization of PFN1. CCK-8 and flow cytometry were performed to validate biological function. A total of 9 LRGs (TRIM28, PPIA, SOD1, RRP1B, IARS2, RB1, PFN1, PRCC, and FABP5) were selected to establish the prognostic signature. Kaplan-Meier survival curves showed that high-risk group patients had a remarkably worse prognosis in the training and validation cohorts. A nomogram was constructed based on LRGs signature and clinical characteristics, and showed excellent predictive power by calibration curve and C-index. Moreover, biological pathways, immunologic status, as well as sensitivity to chemotherapy drugs were different between high- and low-risk groups. Additionally, the hub gene PFN1 is highly expressed in MM, knocking down PFN1 induces cell cycle arrest, suppresses cell proliferation and promotes cell apoptosis. In conclusion, our study revealed that LRGs signature is a promising biomarker for MM that can effectively early distinguish high-risk patients and predict prognosis.

Structural homology of mite profilins to plant profilins is not indicative of allergic cross-reactivity.

Structural and allergenic characterization of mite profilins has not been previously pursued to a similar extent as plant profilins. Here, we describe structures of profilins originating from Tyrophagus putrescentiae (registered allergen Tyr p 36.0101) and Dermatophagoides pteronyssinus (here termed Der p profilin), which are the first structures of profilins from Arachnida. Additionally, the thermal stabilities of mite and plant profilins are compared, suggesting that the high number of cysteine residues in mite profilins may play a role in their increased stability. We also examine the cross-reactivity of plant and mite profilins as well as investigate the relevance of these profilins in mite inhalant allergy. Despite their high structural similarity to other profilins, mite profilins have low sequence identity with plant and human profilins. Subsequently, these mite profilins most likely do not display cross-reactivity with plant profilins. At the same time the profilins have highly conserved poly(l-proline) and actin binding sites.

Correlation between Ki-67 or Profilin-1 Expression Levels, Clinicopathological Characteristics and Postoperative Prognosis in Patients with Bladder Cancer.

BACKGROUND: Bladder cancer is the most common malignancy of the urinary system, and the search for new and reliable biomarkers has important clinical significance for the personalized treatment of bladder cancer. This study aims to explore the correlation between nuclear proliferation antigen (Ki-67) or Profilin-1 (PFN1) levels, clinicopathological characteristics, and postoperative prognosis in patients with bladder cancer. METHODS: Patients with bladder cancer who underwent transurethral resection of bladder cancer tumor in The Fourth Affiliated Hospital of Soochow University, hospital from January 2019 to January 2021 were selected as the study group (n = 60), and patients with benign lesions of bladder cancer during the same period were selected as the control group (n = 60). The expression of Ki-67 and PFN1 in tumor and bladder tissues of the two groups was analyzed. Ki-67 recorded the patient's pathological parameters and calculated the patient's postoperative prognosis. The correlation between Ki-67 and PFN1 expression levels, pathological parameters, and postoperative prognosis was analyzed. RESULTS: The positive expression rates of Ki-67 and PFN1 in the study group were 63.33% and 73.33%, respectively, which were significantly higher than the positive expression rates in the control group (χ2 = 14.803, 17.757, p < 0.001). The positive expression rates of Ki-67 and PFN1 were related to histological grade, clinical stage, infiltration, and lymph node metastasis, and the differences were statistically significant (p < 0.05). Bladder cancer patients with non muscle-invasive bladder cancer (NMIBC), high-grade histological grade, Ta~T1 clinical stage, invasive, and lymph node metastasis have a higher Ki-67 positive expression rate than bladder cancer patients with muscle-invasive bladder cancer (MIBC), low-grade histological grade, T2~T4, non-invasive, and no lymph node metastasis. The high expression level of Ki-67 has little relationship with gender, age, tumor diameter, and vascular invasion (p > 0.05). The survival time and three-year survival rate of the Ki-67 positive expression group were significantly lower than those of the Ki-67 negative expression group (p < 0.05). The survival time and three-year survival rate of the PFN1 positive expression group were significantly lower than those of the PFN1 negative expression group (p < 0.05). CONCLUSION: The positive expression rates of Ki-67 and PFN1 in bladder tumor tissue are significantly higher than those in bladder tissue, and pathological pattern, histological grade, clinical stage, infiltration, and lymph node metastasis are related to the positive expression rates of Ki-67 and PFN1, and different genders, ages, tumors diameter and vascular invasion are not related to the positive expression rates of Ki-67 and PFN1. The survival time and three-year survival rates of bladder cancer patients with Ki-67 positive and PFN1 positive expression are shorter.

Global acetylome profiling indicates EPA impedes but OA promotes prostate cancer motility through altered acetylation of PFN1 and FLNA.

Prostate cancer (PCa) is one of the leading causes of cancer morbidity and mortality in men. Metastasis is the main cause of PCa-associated death. Recent evidence indicated a significant reduction in PCa mortality associated with higher ω-3 polyunsaturated fatty acids (PUFAs) consumption. However, the underlying mechanisms remained elusive. In this study, we applied global acetylome profiling to study the effect of fatty acids treatment. Results indicated that oleic acid (OA, monounsaturated fatty acid, MUFA, 100 µM) elevates while EPA (eicosapentaenoic acid, 100 µM) reduces the acetyl-CoA level, which alters the global acetylome. After treatment, two crucial cell motility regulators, PFN1 and FLNA, were found with altered acetylation levels. OA increased the acetylation of PFN1 and FLNA, whereas EPA decreased PFN1 acetylation level. Furthermore, OA promotes while EPA inhibits PCa migration and invasion. Immunofluorescence assay indicated that EPA impedes the formation of lamellipodia or filopodia through reduced localization of PFN1 and FLNA to the leading edge of cells. Therefore, perturbed acetylome may be one critical step in fatty acid-affected cancer cell motility. This study provides some new insights into the response of ω-3 PUFAs treatment and a better understanding of cancer cell migration and invasion modulation.

Combining bulk and scRNA-seq to explore the molecular mechanisms governing the distinct efferocytosis activities of a macrophage subpopulation in PDAC.

Pancreatic ductal adenocarcinoma (PDAC), a very aggressive tumour, is currently the third leading cause of cancer-related deaths. Unfortunately, many patients face the issue of inoperability at the diagnostic phase leading to a quite dismal prognosis. The onset of metastatic processes has a crucial role in the elevated mortality rates linked to PDAC. Individuals with metastatic advances receive only palliative therapy and have a grim prognosis. It is essential to carefully analyse the intricacies of the metastatic process to enhance the prognosis for individuals with PDAC. Malignancy development is greatly impacted by the process of macrophage efferocytosis. Our current knowledge about the complete range of macrophage efferocytosis activities in PDAC and their intricate interactions with tumour cells is still restricted. This work aims to resolve communication gaps and pinpoint the essential transcription factor that is vital in the immunological response of macrophage populations. We analysed eight PDAC tissue samples sourced from the gene expression omnibus. We utilized several software packages such as Seurat, DoubletFinder, Harmony, Pi, GSVA, CellChat and Monocle from R software together with pySCENIC from Python, to analyse the single-cell RNA sequencing (scRNA-seq) data collected from the PDAC samples. This study involved the analysis of a comprehensive sample of 22,124 cells, which were classified into distinct cell types. These cell types encompassed endothelial and epithelial cells, PDAC cells, as well as various immune cells, including CD4+ T cells, CD8+ T cells, NK cells, B cells, plasma cells, mast cells, monocytes, DC cells and different subtypes of macrophages, namely C0 macrophage TGM2+, C1 macrophage PFN1+, C2 macrophage GAS6+ and C3 macrophage APOC3+. The differentiation between tumour cells and epithelial cells was achieved by the implementation of CopyKat analysis, resulting in the detection and categorization of 1941 PDAC cells. The amplification/deletion patterns observed in PDAC cells on many chromosomes differ significantly from those observed in epithelial cells. The study of Pseudotime Trajectories demonstrated that the C0 macrophage subtype expressing TGM2+ had the lowest level of differentiation. Additionally, the examination of gene set scores related to efferocytosis suggested that this subtype displayed higher activity during the efferocytosis process compared to other subtypes. The most active transcription factors for each macrophage subtype were identified as BACH1, NFE2, TEAD4 and ARID3A. In conclusion, the examination of human PDAC tissue samples using immunofluorescence analysis demonstrated the co-localization of CD68 and CD11b within regions exhibiting the presence of keratin (KRT) and alpha-smooth muscle actin (α-SMA). This observation implies a spatial association between macrophages, fibroblasts, and epithelial cells. There is variation in the expression of efferocytosis-associated genes between C0 macrophage TGM2+ and other macrophage cell types. This observation implies that the diversity of macrophage cells might potentially influence the metastatic advancement of PDAC. Moreover, the central transcription factor of different macrophage subtypes offers a promising opportunity for targeted immunotherapy in the treatment of PDAC.

Oncogenic circ-SLC16A1 promotes progression of non-small cell lung cancer via regulation of the miR-1287-5p/profilin 2 axis.

BACKGROUND: Circular RNAs (circRNAs) are single-stranded RNAs with covalently closed structures that have been implicated in cancer progression. However, the regulatory mechanisms remain largely unclear. So, the aim of this study was to reveal the role and regulatory mechanisms of circ-SLC16A1. METHODS: In this study, next-generation sequencing was used to identify abnormally expressed circRNAs between cancerous and para-carcinoma tissues. Fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction were performed to assess the expression patterns of circ-solute carrier family 16 member 1 (SLC16A1) in non-small cell lung cancer (NSCLC) cells and tissue specimens. The dual-luciferase reporter assay was utilized to identify downstream targets of circ-SLC16A1. Transwell migration, wound healing, 5-ethynyl-2'-deoxyuridine incorporation, cell counting, and colony formation assays were conducted to assess the proliferation and migration of NSCLC cells. A mouse tumor xenograft model was employed to determine the roles of circ-SLC16A1 in NSCLC progression and metastasis in vivo. RESULTS: The results found that circ-SLC16A1 was upregulated in NSCLC cells and tissues. Downregulation of circ-SLC16A1 inhibited tumor growth by reducing proliferation, lung metastasis, and lymphatic metastasis of NSCLC cells, and arrested the cell cycle in the G1 phase. Also, silencing of circ-SLC16A1 promoted apoptosis of NSCLC cells. The results of bioinformatics analysis and the dual-luciferase reporter assay confirmed that microRNA (miR)-1287-5p and profilin 2 (PFN2) are downstream targets of circ-SLC16A1. PFN2 overexpression or circ-SLC16A1 inhibition restored proliferation and migration of NSCLC cells after silencing of circ-SLC16A1. PFN2 overexpression restored migration and proliferation of NSCLC cells post miR-1287-5p overexpression. CONCLUSIONS: Collectively, these findings show that miR-1287-5p/PFN2 signaling was associated with downregulation of circ-SLC16A1 and reduced invasion and proliferation of NSCLC cells. So, circ-SLC16A1 is identified as a mediator of multiple pro-oncogenic signaling pathways in NSCLC and can be targeted to suppress tumor progression.

OptoProfilin: A Single Component Biosensor of Applied Cellular Stress.

The actin cytoskeleton is a biosensor of cellular stress and a potential prognosticator of human disease. In particular, aberrant cytoskeletal structures such as stress granules formed in response to energetic and oxidative stress are closely linked to ageing, cancer, cardiovascular disease, and viral infection. Whether these cytoskeletal phenomena can be harnessed for the development of biosensors for cytoskeletal dysfunction and, by extension, disease progression, remains an open question. In this work, we describe the design and development of an optogenetic iteration of profilin, an actin monomer binding protein with critical functions in cytoskeletal dynamics. We demonstrate that this optically activated profilin ('OptoProfilin') can act as an optically triggered biosensor of applied cellular stress in select immortalized cell lines. Notably, OptoProfilin is a single component biosensor, likely increasing its utility for experimentalists. While a large body of preexisting work closely links profilin activity with cellular stress and neurodegenerative disease, this, to our knowledge, is the first example of profilin as an optogenetic biosensor of stress-induced changes in the cytoskeleton.

Actin-binding protein profilin1 is an important determinant of cellular phosphoinositide control.

Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.

Long noncoding RNA RMRP ameliorates doxorubicin-induced apoptosis by interacting with PFN1 in a P53-Dependent manner.

Doxorubicin (DOX) often causes acute or chronic cardiotoxicity during its application. LncRNA RMRP has been reported to be associated with several biological processes, such as cartilage-hair hypoplasia, but the relationship between RMRP and DOX-induced cardiotoxicity and chronic heart failure remains obscure. To test this hypothesis, GSE124401 and GSE149870 were processed for bioinformatics, and differentially expressed RMRP was then verified in the peripheral blood of 21 patients with heart failure compared with 7 controls. For in vitro validation, we used AC16 and HEK-293T cells. qPCR was used to detect the mRNA expression levels. The degree of apoptosis was detected by Western blot and TUNEL staining. Furthermore, the interaction between RMRP and PFN1 mRNA was verified by dual-luciferase reporter assays. In bioinformatics, RMRP showed significant downregulation, which was verified in clinical samples (p < 0.001) and DOX-treated AC16 models (p < 0.0001). Next, overexpression of RMRP could significantly alleviate DOX-induced apoptosis, and a potential downstream molecule of RMRP, PFN1, was also negatively associated with this change. RESCUE experiments further confirmed that PFN1 could be regulated by RMRP at both the RNA and protein levels, serving as a downstream mediator of RMRP's cardioprotective effects. This interaction was then confirmed to be a direct combination (p < 0.0001). Finally, we found that overexpression of RMRP could inhibit the expression of p53 and its phosphorylation level by suppressing PFN1. In summary, RMRP could exert cardioprotective effects via the PFN1/p53 axis, holding great promise for serving as a therapeutic target and potential biomarker.

Proximity Proteomics Revealed Aberrant mRNA Splicing Elicited by ALS-Linked Profilin-1 Mutants.

Profilin 1 (PFN1) is a cytoskeleton protein that modulates actin dynamics through binding to monomeric actin and polyproline-containing proteins. Mutations in PFN1 have been linked to the pathogenesis of familial amyotrophic lateral sclerosis (ALS). Here, we employed an unbiased proximity labeling strategy in combination with proteomic analysis for proteome-wide profiling of proteins that differentially interact with mutant and wild-type (WT) PFN1 proteins in human cells. We uncovered 11 mRNA splicing proteins that are preferentially enriched in the proximity proteomes of the two ALS-linked PFN1 variants, C71G and M114T, over that of wild-type PFN1. We validated the preferential interactions of the ALS-linked PFN1 variants with two mRNA splicing factors, hnRNPC and U2AF2, by immunoprecipitation, followed with immunoblotting. We also found that the two ALS-linked PFN1 variants promoted the exonization of Alu elements in the mRNAs of MTO1, TCFL5, WRN and POLE genes in human cells. Together, we showed that the two ALS-linked PFN1 variants interacted preferentially with mRNA splicing proteins, which elicited aberrant exonization of the Alu elements in mRNAs. Thus, our work provided pivotal insights into the perturbations of ALS-linked PFN1 variants in RNA biology and their potential contributions to ALS pathology.

ELAVL4 promotes the tumorigenesis of small cell lung cancer by stabilizing LncRNA LYPLAL1-DT and enhancing profilin 2 activation.

Small cell lung cancer (SCLC) is one of the most malignant tumors that has an extremely poor prognosis. RNA-binding protein (RBP) and long noncoding RNA (lncRNA) have been shown to be key regulators during tumorigenesis as well as lung tumor progression. However, the role of RBP ELAVL4 and lncRNA LYPLAL1-DT in SCLC remains unclear. In this study, we verified that lncRNA LYPLAL1-DT acts as an SCLC oncogenic lncRNA and was confirmed in vitro and in vivo. Mechanistically, LYPLAL1-DT negatively regulates the expression of miR-204-5p, leading to the upregulation of PFN2, thus, promoting SCLC cell proliferation, migration, and invasion. ELAVL4 has been shown to enhance the stability of LYPLAL1-DT and PFN2 mRNA. Our study reveals a regulatory pathway, where ELAVL4 stabilizes PFN2 and LYPLAL1-DT with the latter further increasing PFN2 expression by blocking the action of miR-204-5p. Upregulated PFN2 ultimately promotes tumorigenesis and invasion in SCLC. These findings provide novel prognostic indicators as well as promising new therapeutic targets for SCLC.

Genetic Screening of ZNF687 and PFN1 in a Paget's Disease of Bone Cohort Indicates an Important Role for the Nuclear Localization Signal of ZNF687.

Paget's disease of bone (PDB) is a common, late-onset bone disorder, characterized by focal increases of bone turnover that can result in bone lesions. Heterozygous pathogenic variants in the Sequestosome 1 (SQSTM1) gene are found to be the main genetic cause of PDB. More recently, PFN1 and ZNF687 have been identified as causal genes in patients with a severe, early-onset, polyostotic form of PDB, and an increased likelihood to develop giant cell tumors. In our study, we screened the coding regions of PFN1 and ZNF687 in a Belgian PDB cohort (n = 188). In the PFN1 gene, no variants could be identified, supporting the observation that variants in this gene are extremely rare in PDB. However, we identified 3 non-synonymous coding variants in ZNF687. Interestingly, two of these rare variants (p.Pro937His and p.Arg939Cys) were clustering in the nuclear localization signal of the encoded ZNF687 protein, also harboring the p.Pro937Arg variant, a previously reported disease-causing variant. In conclusion, our findings support the involvement of genetic variation in ZNF687 in the pathogenesis of classical PDB, thereby expanding its mutational spectrum.

Hypoxia-based classification and prognostic signature for clinical management of hepatocellular carcinoma.

OBJECTIVE: Intratumoral hypoxia is an essential feature of hepatocellular carcinoma (HCC). Herein, we investigated the hypoxia-based heterogeneity and relevant clinical implication in HCC. METHODS: Three HCC cohorts: TCGA-LIHC, LICA-FR, and LIRI-JP were retrospectively gathered. Consensus clustering analysis was utilized for hypoxia-based classification based upon transcriptome of hypoxia genes. Through LASSO algorithm, a hypoxia-relevant prognostic signature was built. Immunotherapeutic response was inferred through analyzing immune checkpoints, T cell inflamed score, TIDE score, and TMB score. RNF145 expression was measured in normoxic or hypoxic HCC cells. In RNF145-knockout cells, CCK-8, TUNEL, and scratch tests were implemented. RESULTS: HCC patients were classified into two hypoxia subtypes, with more advanced stages and poorer prognosis in cluster2 than cluster1. The heterogeneity in tumor infiltrating immune cells and genetic mutation was found between subtypes. The hypoxia-relevant prognostic model was proposed, composed of ANLN, CBX2, DLGAP5, FBLN2, FTCD, HMOX1, IGLV1-44, IL33, LCAT, LPCAT1, MKI67, PFN2, RNF145, S100A9, and SPP1). It was predicted that high-risk patients presented worse prognosis with an independent and reliable manner. Based upon high expression of immune checkpoints (CD209, CTLA4, HAVCR2, SIRPA, TNFRSF18, TNFRSF4, and TNFRSF9), high T cell inflamed score, low TIDE score and high TMB score, high-risk patients might respond to immunotherapy. Experimental validation showed that RNF145 was upregulated in hypoxic HCC cells, RNF145 knockdown attenuated proliferation and migration, but aggravated apoptosis in HCC cells. CONCLUSION: Altogether, the hypoxia-based classification and prognostic signature might be useful for prognostication and guiding treatment of HCC.

Vascular endothelial profilin-1 drives a protumorigenic tumor microenvironment and tumor progression in renal cancer.

Overexpression of actin-binding protein profilin-1 (Pfn1) correlates with advanced disease features and adverse clinical outcome of patients with clear cell renal carcinoma, the most prevalent form of renal cancer. We previously reported that Pfn1 is predominantly overexpressed in tumor-associated vascular endothelial cells in human clear cell renal carcinoma. In this study, we combined in vivo strategies involving endothelial cell-specific depletion and overexpression of Pfn1 to demonstrate a role of vascular endothelial Pfn1 in promoting tumorigenicity and enabling progressive growth and metastasis of renal carcinoma cells in a syngeneic orthotopic mouse model of kidney cancer. We established an important role of endothelial Pfn1 in tumor angiogenesis and further identified endothelial Pfn1-dependent regulation of several pro- (VEGF, SERPINE1, CCL2) and anti-angiogenic factors (platelet factor 4) in vivo. Endothelial Pfn1 overexpression increases tumor infiltration by macrophages and concomitantly diminishes tumor infiltration by T cells including CD8+ T cells in vivo, correlating with the pattern of endothelial Pfn1-dependent changes in tumor abundance of several prominent immunomodulatory cytokines. These data were also corroborated by multiplexed quantitative immunohistochemistry and immune deconvolution analyses of RNA-seq data of clinical samples. Guided by Upstream Regulator Analysis of tumor transcriptome data, we further established endothelial Pfn1-induced Hif1α elevation and suppression of STAT1 activation. In conclusion, this study demonstrates for the first time a direct causal relationship between vascular endothelial Pfn1 dysregulation, immunosuppressive tumor microenvironment, and disease progression with mechanistic insights in kidney cancer. Our study also provides a conceptual basis for targeting Pfn1 for therapeutic benefit in kidney cancer.

Hydrogen peroxide oxidation modifies the structural properties and allergenicity of the bee pollen allergen profilin.

Bee pollen is a byproduct of pollination, which is a necessary process to produce foods. However, bee pollen can induce significant food-borne allergies. We previously identified a bee pollen-derived pan-allergen in the profilin family, Bra c p. Herein, we aimed to reduce Bra c p allergenicity via protein oxidation with hydrogen peroxide and explore the changes induced. Ion-mobility mass spectrometry revealed aggregation of the oxidized product; we also found irreversible sulfonation of the free sulfhydryl group of the Bra c p Cys98 residue to a more stable cysteine derivative. A significant proportion of the α-helices in Bra c p were transformed into ß-sheets after oxidation, masking the antigenic epitopes. An immunoassay demonstrated that the IgE-binding affinity of Bra c p was decreased in vitro after oxidation. To our knowledge, this is the first report describing the application of protein oxidation to reduce the allergenicity of profilin family member in foods.

Profilin1 Regulates Trophoblast Invasion and Macrophage Differentiation.

Unexplained recurrent spontaneous abortion (URSA) has been associated with the dysfunction of trophoblasts and decidual macrophages. Current evidence suggests that profilin1 (PFN1) plays an important role in many biological processes. However, little is known about whether PFN1 is related to URSA. Herein, the location of PFN1 was detected by immunohistochemistry, and the level of PFN1 was detected by quantitative real-time PCR, Western blot analysis, and immunohistochemistry. The proliferation of trophoblasts was detected by CCK8 and 5-ethynyl-2'-deoxyuridine assays, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays were used to detect apoptosis of trophoblasts. The migration and invasion ability of trophoblasts was assessed by using the wound-healing test and transwell test. Polarization of macrophages was detected in macrophages cultured in trophoblast conditioned medium. PFN1 expression was observed in cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts and was decreased in the villous tissue of patients with URSA. The migration and invasion ability and cell viability of trophoblastic cell lines that underwent PFN1 knockdown significantly decreased, and apoptosis increased. Opposite findings were observed after the overexpression of PFN1 in trophoblastic cells. In addition, PFN1 could regulate trophoblast function through phosphatidylinositol 3-kinase/AKT signal transduction rather than mitogen-activated protein kinase signaling pathways. Finally, knockdown of PFN1 in trophoblasts promoted tumor necrosis factor-α secretion to induce macrophage polarization to M1 phenotype, mediated by the NF-κB signaling pathway. These findings indicate that PFN1 has a broad therapeutic potential for patients with URSA.

A novel Alzheimer's disease prognostic signature: identification and analysis of glutamine metabolism genes in immunogenicity and immunotherapy efficacy.

Alzheimer's disease (AD) is characterized as a distinct onset and progression of cognitive and functional decline associated with age, as well as a specific neuropathology. It has been discovered that glutamine (Gln) metabolism plays a crucial role in cancer. However, a full investigation of its role in Alzheimer's disease is still missing. This study intended to find and confirm potential Gln-related genes associated with AD using bioinformatics analysis. The discovery of GlnMgs was made possible by the intersection of the WGCNA test and 26 Gln-metabolism genes (GlnMgs). GlnMgs' putative biological functions and pathways were identified using GSVA. The LASSO method was then used to identify the hub genes as well as the diagnostic efficiency of the four GlnMgs in identifying AD. The association between hub GlnMgs and clinical characteristics was also studied. Finally, the GSE63060 was utilized to confirm the levels of expression of the four GlnMgs. Four GlnMgs were discovered (ATP5H, NDUFAB1, PFN2, and SPHKAP). For biological function analysis, cell fate specification, atrioventricular canal development, and neuron fate specification were emphasized. The diagnostic ability of the four GlnMgs in differentiating AD exhibited a good value. This study discovered four GlnMgs that are linked to AD. They shed light on potential new biomarkers for AD and tracking its progression.

Downregulation of the Rho GTPase pathway abrogates resistance to ionizing radiation in wild-type p53 glioblastoma by suppressing DNA repair mechanisms.

Glioblastoma (GBM), the most common aggressive brain tumor, is characterized by rapid cellular infiltration and is routinely treated with ionizing radiation (IR), but therapeutic resistance inevitably recurs. The actin cytoskeleton of glioblastoma cells provides their high invasiveness, but it remains unclear whether Rho GTPases modulate DNA damage repair and therapeutic sensitivity. Here, we irradiated glioblastoma cells with different p53 status and explored the effects of Rho pathway inhibition to elucidate how actin cytoskeleton disruption affects the DNA damage response and repair pathways. p53-wild-type and p53-mutant cells were subjected to Rho GTPase pathway modulation by treatment with C3 toxin; knockdown of mDia-1, PFN1 and MYPT1; or treatment with F-actin polymerization inhibitors. Rho inhibition increased the sensitivity of glioma cells to IR by increasing the number of DNA double-strand breaks and delaying DNA repair by nonhomologous end-joining in p53-wild-type cells. p53 knockdown reversed this phenotype by reducing p21 expression and Rho signaling activity, whereas reactivation of p53 in p53-mutant cells by treatment with PRIMA-1 reversed these effects. The interdependence between p53 and Rho is based on nuclear p53 translocation facilitated by G-actin and enhanced by IR. Isolated IR-resistant p53-wild-type cells showed an altered morphology and increased stress fiber formation: inhibition of Rho or actin polymerization decreased cell viability in a p53-dependent manner and reversed the resistance phenotype. p53 silencing reversed the Rho inhibition-induced sensitization of IR-resistant cells. Rho inhibition also impaired the repair of IR-damaged DNA in 3D spheroid models. Rho GTPase activity and actin cytoskeleton dynamics are sensitive targets for the reversal of acquired resistance in GBM tumors with wild-type p53.

Toxoplasma gondii microneme protein MIC3 induces macrophage TNF-α production and Ly6C expression via TLR11/MyD88 pathway.

Toxoplasma gondii is the most successful parasite worldwide. It is of great interest to understand how T. gondii induce different immune responses in different hosts. In this study, we found that a peptide of T. gondii microneme protein MIC3 induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression in mouse macrophage RAW264.7 cells. MyD88 inhibition, small interfering RNA against Tlr11 and CRISPR/Cas9-mediated knock-out of Tlr11 all reduced MIC3-induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression. Additionally, we determined the location of MIC3 peptide in mouse macrophages using immunofluorescence. MIC3 could both adhere to the cell membrane of mouse macrophages and enter the cells. These results suggest that MIC3 triggered the immune responses in mouse macrophages via TLR11/MyD88/NF-κB pathway. It is known that human macrophages lacking TLR11. We predicted that the immune responses induced by MIC3 in human macrophages were significantly different from those in mouse macrophages. As expected, MIC3 peptide failed to induce TNF-α expression, iNOS expression and NF-κB phosphorylation in human THP-1 derived macrophages. MIC3 induced macrophage immune responses via TLR11. Intriguingly, the amino acid sequence of MIC3 is completely different from the well-known TLR11 ligand profilin, which generates a potent IL-12p40, TNF-α and IL-6 response. In marked contrast to profilin, MIC3 could not induce IL-12p40 expression in both mouse RAW264.7 cells and human THP-1 derived macrophages. Furthermore, the simulated tertiary structure of MIC3 peptide shows poor similarity with the crystal structure of profilin, suggesting that MIC3 might be a different ligand from profilin. These findings about MIC3 and TLR11 will provide us with important insights into the pathogenesis of toxoplasmosis and coevolution during host-parasite interaction.